A characteristic of the interiors of all cells is the high total concentration of macromolecules they contain. Such media are termed ‘crowded’ rather than ‘concentrated’ because, in general, no single macromolecular species occurs at high concentration but, taken together, show a volume occupancy of 20-30% of a given specific volume with a concentration close to 1 kg/l. Thus, these values define an approximate range of 200-300 g/l to be considered when using physical theory to calculate the consequences of crowding inside cells. As pointed out by Ellis (2001) and Minton (2000) crowding by macromolecules has both thermodynamic and kinetic effects on the properties of other macromolecules that are not generally appreciated (Minton 2000, Ellis 2001). Biological macromolecules such as enzymes have evolved to function inside such crowded environments. For example, the total concentration of protein and RNA inside bacteria like E. coli is in the range of 300-400 g/l. Macromolecular crowding is also termed the excluded volume effect, because its most basic characteristic is the mutual impenetrability of all solute molecules. This nonspecific steric repulsion is always present, regardless of any other attractive or repulsive interactions that might occur between the solute molecules. Thus, crowding cannot be avoided and is a hallmark of the intracellular milieu of all carbon-based life-forms on earth (reviewed in Ellis 2001). These resulting effects are so large that authorities in the field state that many estimates of enzyme catalyzed reaction rates and equilibria made with uncrowded solutions in the test tube differ by orders of magnitude from those of the same reactions operating under crowded conditions within cells (Ellis 2001).
Surprisingly, biochemists commonly study enzymatic reactions in solutions with a total macromolecular concentration of 1-10 g/l or less, in which crowding is negligible. In particular, all biochemical and enzymatic reactions relating to the amplification of RNA and DNA for diagnostic, scientific and therapeutic purposes are done in aqueous media under conditions that do not reflect the crowded environment of the organism they have been originally derived from. Of note, transcriptase and polymerases used in these systems are derived from thermophilic bacteria, most prominently Thermus aquaticus (Taq polymerase). These thermophilic bacteria reside and survive in hot pools and the enzymes derived from these organisms are employed under similar temperatures. The similarity of working conditions ends here, since these enzymes are put to work in aqueous solutions that do not emulate the crowded conditions in the bacteria they were originally derived from. Taq polymerase is a non-replicative type of DNA polymerase with an inherently low processivity compared to replicative type polymerases (T4 polymerase) which depend on specific peptides for their high processivity in vivo. Hence, to make Taq highly processive, it has to be “coaxed” to polymerise DNA in vitro for a detectable yield of PCR product that does not normally happen in vivo. In physiological conditions it was suggested that macromolecular crowding could enhance and/or maintain the integrity and/or stability of the DNA-Polymerase complex (Zimmermann and Harrison, 1987), although this was suggested only in reference to a T4 E. coli DNA polymerase binding to its target DNA at room temperature. Non-crowding approaches to increase processivity have revolved around complex procedures like engineering recombinant constructs to synthesise peptides that function as ‘processivity factors’ to enhance the binding of the enzyme to the target DNA in a “sliding clamp” mechanism which only partially resembles the normal physiology. Certain non-replicative polymerases such as the T7 DNA polymerase may have their processivity enhanced in the presence of proteins like thioredoxin, with the resultant T7-thioredoxin complex being able to function like the sliding clamp processor of replicative polymerases. A recent genetically engineered construct was suggested for synthesizing a peptide to enhance the binding between the polymerase and its target DNA (Wang et al, 2004) in a non-sequence dependent manner.
Though the current methods have been suggested to be effective, they do not address the underlying flaw of not observing that macromolecular crowding is an inherent physiological requirement for a highly processive DNA and RNA polymerization in vivo and hence in vitro.
Another aspect of in vitro DNA polymerization (as in a PCR) that has, not been currently addressed is the degree of fidelity of the polymerizing enzyme. This refers to the ability of the enzyme to incorporate the correct nucleotides complementary to the target sequence, so that minimal error is found in the final product. Current measures to enhance fidelity involve using thermostable polymerases armed with proof-reading properties or having been engineered to carry them. This slows down the processivity of the enzyme automatically. Hence, ensuring simultaneous high processivity and high fidelity is still eluding current molecular biology techniques. Again, physiological in vivo DNA synthesis occurs in a highly crowded environment within the nucleus. In this situation, it has been theorized and biophysically shown that crowding favors correct DNA base-pair matching (the match-making effect) under physiological conditions (Goobes et al, 2003).
From a biophysical standpoint, the absence of crowding conditions in molecular biology techniques must lead to a variety of deficiencies when performing these extremely widely used procedures
1) PCR—Polymerase Chain Reaction
                without chaperones and protecting macromolecular crowders, folding of enzymes is compromised and they are, albeit sturdy, subjected to increased hydrolysis and decay towards the end of PCR cycles;        the polymerizing enzymes are not optimally folded and perform poorly under high temperatures after several cycles, leading to poor yield (enzymatic exhaustion) and copying mistakes (reduced fidelity);        the DNA-enzyme, DNA/DNA, RNA/DNA, RNA/RNA and related enzyme/protein complexes are less stable leading to suboptimal enzyme substrate kinetics compared to that in vivo;        with more stringent conditions such as low copy number of target DNA, the detection ability with routine PCR conditions often fails;        under stringent conditions of salt and temperature (large cycle number), the physical limits of the enzyme activity with regard to stability and processivity, are soon reached and hence the enzyme often fails to synthesize a complete complementary copy of the target. This results in production of too short PCR products;        the non-specific binding of primers to target is another problem with conventional PCRs that needs to be addressed by methods that select only the correctly bound primer-target duplex for further extension.        
Prior art has tried to remedy above deficiencies by either modifying the amplifying enzymes (purification, recombinant expression, site directed mutagenesis (Gottlieb et al 1990 that describes a type 1 UL42 gene product which is a subunit of DNA polymerase that functions to increase processivity and WO0192501), or elaborate polymerization protocols (Wang et al. 2000). Only two approaches are documented that deal with the modification of the PCR reaction medium as such.
(A) The first approach is based on the principle of water structuring and preferential hydration due to compatible solutes, also known as osmolytes (U.S. Pat. Nos. 6,114,150A; 6,428,986; 6,300,073). This family comprises of several low molecular weight compounds that may be derived from carbohydrates (sucrose, trehalose), aminoacids (betaine, proline) or ectoines (homoectoine), all of which belong to the class of compatible solutes. The addition of the small molecule, trehalose, has been shown to be beneficial for PCR reactions with difficult target templates to perform leading to a higher yield. Trehalose, a sugar that is only found in certain lower organisms and actively synthesized in Th. aquaticus, is the original source for in vitro DNA polymerase (Taq polymerase). Thus, it is a natural component of the interior of the bacterial cell and known to belong to the group of compatible solutes (Spiess et al. 2002). that help these organisms to withstand extreme physico-chemical environments. However, trehalose with a molecular weight of 342 Da is far from being a macromolecule and thus, even in huge amounts cannot substitute them. In addition, a small but significant decrease in melting temperature of DNA or RNA hybrids in the presence of trehalose changes basic parameters of a typical PCR reaction as such. But this is supposedly the mechanism by which trehalose and the other like-compounds function, i.e by reducing the melting temperature of the target DNA. They have been mostly effective only when the target DNA or RNA has a significant secondary structure which could be linearized by such compounds that reduce their melting temperatures. However, this in itself limits the utility of these compounds to only a small sector of their applications on PCR.
(B) The second approach is also based on the concept of structuring water using engineered nanoparticles. The addition of nanoparticles (Neowater©) to the PCR mix as given in WO03053647. The nanoparticles are derived from metallic materials such as BaTiO3, Ba2F9O12, WO3 in a top-down approach by breaking down 10 μm particles ending up in particles of a size distribution of 5-50 nm which retain their crystalline structure. The application of nanoparticles described in WO03053647 is claimed to work on the basis of structuring water and propagating alignment of water molecules. It is difficult to conceive how water molecules should be able maintain a structured state under PCR-typical high temperatures (close to boiling point of water) with its typical excess of Brownian molecular movements.
Chebotareva et al., 2004 reviewed the basic mechanisms by which the low molecular weight solutes (compatible solutes; osmolytes) effected a molecular crowding phenomenon which differs from the principle of macromolecular crowding and is the essential platform of our invention. Basically, molecular crowders work by structuring water (kosmotropes), whilst the macromolecular crowders function by excluding volume due to non-specific steric repulsive interactions. The implications of this basic fact are significant: while one has to use hundreds of milligrams of cosolutes per milliliter of solvent, macromolecules will be effective in micrograms to a few milligrams per ml of the solvent. This keeps the reaction conditions unchanged in terms of viscosity, pH and electrolyte concentrations which are key determinants of any biological enzymatic reaction. In fact, osmolytes in high concentrations can act as “molecular brakes” and reduce the rate of enzymatic reactions significantly (Spiess et al. 2004). Earlier work before the PCR era has shown that the addition of the macromolecules Ficoll 70 and Dextran T 70 to polymerase reactions from E. coli derived enzymes at 37° C. improved DNA enzyme binding and resulted in a longer survival of polymerase activity (Zimmermann & Harrison 1987). However, Ficoll 70 used alone was not resistant to the usual PCR temperatures. Further, Wenner and Bloomfield, 1999, have studied the possible crowding effect of Ficoll 70 of EcoRV cleavage. However, the results indicated that Ficoll had little effect on EcoRV reaction velocity.
2) Reverse Transcriptase Reaction
This is basically the most crucial step in all amplification procedures in order to create cDNA from extracted total mRNA. The extraction yield of this material is usually very limited, in particular if only a small collection of cells from needle biopsies of tissue or laser dissected portions from histological sections or forensic samples (saliva, fingerprints, blood, sperm) are available. The faithful and specific reverse transcription process will not only rule the amount and yield but also specificity and number of faulty copies generated (fidelity). Basically, the same shortcomings as listed for PCR apply. The current remedial action in prior art is to create high quality reverse transcripts of total mRNA are based on i) creation of novel and modified reverse transcriptases ii) modifications on the amplification procedures as such (template switching). With the advent of automation in RT-PCR, it has made the utility of enhancing this step of cDNA synthesis all the more crucial for obtaining a reasonable yield of second strand DNA downstream. With some recent reports of miniaturization of PCR using MEMS technology, application of principles to improve the environmental conditions of both reverse transcription for first strand cDNA synthesis and ds-cDNA has assumed prime importance.
However as yet, there are no reports of a combined approach to enhance simultaneously both the first and second strand cDNA syntheses.